Ion-exchange chromatography (IEC) is the most widely used chromatographic tool in protein purification schemes, and is performed either as a high- or low pressure technique. Ion-exchange sorbents are usually based on inorganic silica beads or polymeric particles, which are provided with surface cationic or anionic groups capable of interacting electrostatically with ionic species of opposite charge. The retention of a protein will thus depend on the charge status of the protein, the amount of interaction sites available on the sorbent, and on the strength of the individual interactions. Displacement of the analyte is normally achieved by competitively increasing the mobile-phase ionic strength, and relatively high salt concentrations are often required in order to elute even moderately retained proteins [Deutscher, M. P. (Ed.), Guide to Protein Purification (Meth. Enzymol., Vol. 182), Academic Press, 1990]. Even if many proteins can withstand high salt concentrations without severe denaturation, there are usually other practical limits to the ionic strength of the eluent, such as “salting out”, or the desire to produce a separated sample in a relatively low saline buffer to avoid extensive dialysis of the solute after the separation. An ideal ion-exchange sorbent should also be free from non-specific interaction sites, possess an internal porosity that allows even large proteins to enter, and have a permeability and a mechanical rigidity sufficient for operation at high flow rates (Müller, W., J Chromatogr., 1990, 510, 133-140).
Accordingly, there is a need for an improved ion-exchange chromatography method which does not require elution using high salt concentrations, and which fulfills as many of the above mentioned criteria regarding ideal ion-exchange sorbents as possible.
Because of tie significance of bioseparations both in industry and in research, there is a never ending search for novel stationary phases that can fulfill up-coming requirements. In particular, there is an interest in new separation modes, which can provide selectivities that are orthogonal to existing techniques. In 1992, Svec and Fréchet pioneered a novel kind of separation media consisting of a rigid macroporous monoliths polymerized in-situ within the confines of a chromatographic column (Svec, F.; Fréchet, J. M. J. Anal. Chem. 1992, 64, 830-832). The porous properties of these monoliths can be easily controlled during their synthesis (Viklund, C.; Svec, F.; Fréchet J. M. J.; Irgum, K. Chem. Mater. 1996, 8, 744-750; Viklund, C.; Ponten, E.; Glad, B.; Irgum, K. Svec, F.; Hörstedt, P. Chem. Mater. 1997, 9, 463-471), which makes it possible to design materials suitable for a particular bioseparation application. For example, poly(glycidyl methacrylate-co-ethylene dimethacrylate) monoliths modified to contain diethylamine functional groups have been used in anion exchange mode for the separation of proteins (Svec, F.; Fréchet, J. M. J., Anal. Chem. 1992, 64, 830-832; Svec, F.; Fréchet, J. M. J., J. Chromatogr. A. 1995b, 702, 89-95). Poly(styrene-co-divinylbenzene) monolithic columns have been successfully used for the fast separation of proteins in the reversed phase mode (Wang, Q. C.; Svec, F.; Fréchet, J. M. J. Anal. Chem. 1993, 65, 2243-2248), and it has recently been shown that polyacrylamide based monoliths can be used for the rapid separation of proteins in the hydrodynamic interaction mode when butyl methacrylate is included in tie polymerization mold (Xie, S.; Svec, F.; Fréchet, J. M. J., J. Chromatogr. A., 1997, 775, 65-72). It has also recently been demonstrated that a porous monolithic column grafted with 2-acrylamido-2-methyl-1-propane sulfonic acid can be used for fast cation exchange chromatography of basic proteins (Viklund, C.; Svec, F.; Fréchet, J. M. J.; Irgum, K., Biotechnol. Progr., 1997, 13, 597-600).
Experiments with multiple mode ion exchange chromatographic separations have been done with tandem connection of anion exchange and cation exchange columns in series (E l Rassi Z.; Horwath, C. J. Chromatogr., 1986, 359, 255), and with columns containing mixes of both anion and cation exchange sorbents (Maa, Y. F.; Antia, F.; E l Raasi, Z.; Horwath, C. J. Chromatogr., 1988, 452, 331). Protein separations on mixed cation and anion exchange media have also been carried out on stacks of alternating cation and anion exchange membranes, spaced by neutral membranes (Freitag, R.; Splitt, H; Reif, O.-W., J. Chromatogr., 1996, 728, 129-37).
Chromatographic techniques utilizing zwitterionic moieties in the stationary phase or in the eluent have gained interest since 1981, when Knox and Jurand introduced a technique where quadropolar ion-pairs could be formed when 11-amino undecanoic acid was added to the eluting solution as a “zwitterion-pair agent” (Knox, J. H.; Jurand, J. a) J. Chromatogr. 1981, 203, 85-92; b) J. Chromatogr. 1981, 218, 341-354; J. Chromatogr. 1981, 218, 355-363; J. Chromatogr. 1982, 234, 222-224). This concept was seen to improve the retention of various nucleotides and oligopeptides comprising up to three amino acids in reversed phase chromatography.
Kurganov and co-workers (Kurganov, A. A.; Davankov, V. A.; Unger, K. K. J. Chromatogr. 1991, 548, 207-214) were able to separate acidic and/or basic proteins using a stationary phase which contained both sulfonic acid and quaternary ammonium groups. This mixed mode sorbent, incorrectly termed as being a zwitterionic sorbent in said paper, was prepared by introduction of sulfonic acid and quaternary ammonium groups by sequential chloromethylation, sulfonation and trimethylamination of a styrene layer superficially polymerized onto silica. This will result in ion exchange groups of different charge residing on separate phenyl moieties in the superficial layer, and on p. 212 of said paper, two important aspects are disclosed, that clearly distinguish this mixed mode sorbent from a true zwitterionic sorbent, as disclosed in this document, namely: a) “The result reveal that the ion exchanger contains cationic groups in excess of anion exchange groups” and b) “The peak of lysozyme (last eluting peak) is relatively broad, even at the high flow rate used for elution. It seems that this broadening is due to the mixed-mode interactions between lysozyme and the exchanger.”.
Another example of a mixed mode sorbent is by Nomura and co-workers (Nomura, A.; Yamada, J.; Tsunoda, K. Anal. Chem. 1988, 60, 2509-2512), who reported the preparation of a silica-based HPLC stationary phase onto which amino-containing compounds and carboxy-containing groups, respectively, are independently immobilised. The suitability of this, in reality amphoteric, sorbent for protein separation was also explored. However, no satisfactory results were obtained as some proteins were very strongly adsorbed, and accordingly they were very difficult to elute.
Among the first polymeric carriers intentionally designed to contain a mixture of anion and cation exchange resins were so-called “snake cage resins”, made by polymerizing an acrylic acid “snake” that had been carefully equilibrated with an anion exchange resin (the “cage”) in order to obtain a stoichiometry between the resulting cation and anion exchange sites (Hatch, M. J.; Dillon, A.; Smith, H. B. Ind. Eng. Chem., 1957, 49, 1812). The resulting sorbents were used as “ion retardation resins”, e.g., for desalting of sugar syrup. Although conceptually a 1:1 stoichiometry should be obtained, there were problems manufacturing these sorbents without net ion exchange properties, which are believed to be due to uneven “patchy” distribution of the ion exchange groups in the sorbent (Small, H. Ion Chromatography, Plenum Press: New York, 1989, pp. 133-134). Yu et al. (a) Yu, L. W.; Hartwick, R. A. J. Chromatogr. Sci. 1989, 27, 176-185; b) Yu, L. W.; Floyd, T. R; Hartwich, R. A. J. Chromatogr. Sci. 1986, 24, 177-182) described the preparation of a chemically bonded zwitterionic silica sorbent, and also showed its potential for the separation of nucleotides. No satisfactory results regarding separation of proteins have been reported for this material. In a large number of papers, Hu and co-workers (a) Hu, W.; Takeuchi, T.; Haraguchi, H. Anal. Chem. 1993, 65, 2204-2208; b) Hu, W.; Tao, H.; Haraguchi, H. 1994, 66, 2514-2520) have conducted studies where commercial octadecyl silica columns have been dynamically coated with commercial zwitterionic surfactant reagents. According to their strategy, the zwitterionic surfactant reagents are non-covalently adsorbed to the columns. Using this strategy, simultaneous separation of inorganic cations and anions could be achieved using pure water as the mobile phase. One important reason for using water as the mobile phase is to minimise washing away zwitterionic surfactant during separations. The surfactant has in some cases been included in the eluting solution in order to replace detergents that are continuously washed away from the ODS-column during separations. Hu claims to have separated purified alpha-amylase from saliva on such detergent-modified hydrophobic silica. However, it is important to note that this enzyme passed through the column without retardation as in gel filtration for desalting purposes, and they interpreted their results as being solely due to a size exclusion effect (U.S. Pat. No. 5,589,069; Col. 12, Line 29-31). No other protein was assayed simultaneously, which means that it has not been clarified if the detergent-based dynamic modification method of Hu has the ability of separating different proteins. Finally, it is also important to point out that it is unacceptable in the pharmaceutical industry to use a separation column where part of the column material is leaking out together with compounds that are to be used in pharmaceutical preparations. Polyzwitterions synthesised from zwitterionic monomers have mostly been studied in the field of polymer chemistry because of their fascinating rheological behaviour (Soto, M.; Galin, V. M. Polymer, 1984, 25, 254; Schulz, D. N.; Peiffer, D. G.; Agarwal, P. K.; Larabee, J.; Kaladas, J. J.; Soni, L.; Handwerker, B.; Garner, R. T. Polymer, 1986, 27, 1734-1742; Huglin, M. B.; Rego, J. M. Macromolecules, 1991, 24, 2556-2563) rather than being utilized for chromatographic separation purposes. The most intensively studied class of zwitterion polymers is prepared from monomers with sulfobetaine functionalities, in which the cationic functionality (a quaternary ammonium group) and the anionic functionality (a sulfonate group) are incorporated in close proximity in pendant side chains on the main polymer chain, and it is thus possible to obtain a polymer with zero net charge. It is assumed that the solution behavior of non-crosslinked polyzwitterions is a result of Coulomb interactions between charged groups, and the electrolyte concentration in the surrounding aqueous media will thus have a great influence on the polymer solubility (Soto, M.; Galin, V. M. Polymer, 1984, 25, 254; Schulz, D. N.; Peiffer, D. G.; Agarwal, P. K.; Larabee, J.; Kaladas, J. J.; Soni, L.; Handwerker, B.; Garner, R. T. Polymer, 1986, 27, 1734-1742; Huglin, M. B.; Rego, J. M. Macromolecules, 1991, 24, 2556-2563). In the field of zwitterionic polymeric sorbents, Grote and Schumacher (Grote, M.; Schumacher, U. React. Funct. Polym., 1997, 35, 179-196) have prepared a series of sorbents containing tetrazolinium anion exchange groups, one of which also contained a benzenesulfonic acid group attached to the tetrazolinium ring, thus comprising a zwitterionic group. This sorbent was used for recovery of precious metals.